Recent investigations have identified a novel mutation in the B-RAF gene in the majority of human melanomas but a clear understanding of genetic and biologic events leading to melanoma remains elusive. Experimental investigations on melanoma development have been hampered by two major difficulties: i) the mouse is not an ideal experimental animal for melanoma-related studies because normal melanocytes are differently located in mouse versus human skin; ii) biologically early lesions cannot be obtained from pathologists because of their small size and the necessity for histologic scanning of the entire lesion. Our laboratory has developed a model of human melanoma in which human skin from healthy donors is grafted to immunodeficient mice, followed by intradermal adenovirus vector-mediated expression of three growth factors, bFGF (FGF 2), SCF (c-kit ligand) and ET-3 (endothelin-3) and concomitant irradiation with UVB (ultraviolet light in the B range). After a treatment period of only three to four weeks, highly invasive lesions developed in the human skin grafts that histologically resembled primary melanomas in patients. However, the lesions regressed in the absence of growth factors and cells cultured from the lesions showed limited life span. The first aim proposes to induce tumorigenic melanomas using a novel skin reconstruction model for transduction of genes in melanocytes and stromal fibroblasts. We will test whether melanomas develop when melanocytes are activated by stromal and environmental stimuli, and whether they become immortal and growth factor independent when mutated genes such as mutated B-RAF are introduced. The second aim investigates the mechanisms of transformation of melanocytes to melanoma, testing the hypothesis that the growth factors bFGF, SCF, and ET-3, when produced by stromal fibroblasts, cooperate with UVB to activate critical pathways for growth, survival, and anti-apoptosis. We will focus our investigations on the MAPK, AKT, PKC, and NFKappaB signaling pathways, because we expect that their activation, together with aberrations in defined genes, leads to growth autonomy and limitless proliferative potential. Our unique model allows a systematic dissection of the biological and molecular events leading to human melanoma formation.